Transmission from one host to another is a crucial component of parasite fitness. For some aquatic parasites, transmission occurs via a free-living stage that spends time in the water, awaiting an encounter with a new host. These parasite transmission stages can be impacted by biotic and abiotic factors that influence the parasite’s ability to successfully infect or grow in a new host.

Here we tested whether time spent in the water column and/or exposure to common cyanobacterial toxins impacted parasite transmission stages. More specifically, we tested whether the infectivity, within-host growth, and virulence of the fungal parasite Metschnikowia bicuspidata changed as a result of time spent in the water or from exposure to cyanotoxins in the water column. We exposed parasite transmission spores to different levels of one of two ecologically important cyanotoxins, microcystin-LR and anatoxin-a, and factorially manipulated the amount of time spores were incubated in water. We removed the toxins and used those same spores to infect one genotype of the common lake zooplankton Daphnia dentifera.

We found that cyanotoxins did not impact parasite fitness (infection prevalence and spore yield per infected host) or virulence (host lifetime reproduction and survivorship) at the tested concentrations (10μg/L & 30μg/L). However, we found that spending longer as a transmission spore decreased a spore’s chances for successful infection: spores that were only incubated for 24 hours infected approximately 75% of exposed hosts, whereas spores incubated for 10 days infected less than 50% of exposed hosts.

We also found a negative relationship between the final spore yield from infected hosts and the proportion of hosts that became infected. In treatments where spores spent longer in the water column prior to encountering a host, infection prevalence was lower (indicating lower per spore infectivity), but each infected host yielded more spores at the end of infection. We hypothesize that this pattern may result from intraspecific parasite competition within the host.

Overall, these results suggest that transmission spores of this parasite are not strongly influenced by cyanotoxins in the water column, but that other aspects of spending time in the water strongly influence parasite fitness.

In this study, we used the zooplankton Daphnia dentifera, which is common in stratified lakes in temperate North America (Tessier & Woodruff, 2002). For our experiments, we used the Midland 37 (MID37) genotype, which was isolated from Midland Lake in Greene County, Indiana, and has been used in several prior experiments (e.g., Auld, Hall & Duffy, 2012; Auld et al., 2014). We also used the common fungal parasite Metschnikowia bicuspidata (“Standard” isolate, originally isolated from Baker Lake in Barry County, Michigan). Daphnia become infected by inadvertently consuming transmission spores they encounter in the water column when feeding. By “transmission spore”, we refer to the mature, needle-shaped ascus that contains the ascospore (Metschnikoff, 1884; Codreanu & Codreanu-Balcescu, 1981). After consumption by the host, infection can begin if the needle-shaped spore crosses the gut barrier and is not fought off by a host hemocyte response (Metschnikoff, 1884; Stewart Merrill & Cáceres, 2018). Once infection has taken hold, the fungus replicates within the hemolymph of the host (Stewart Merrill & Cáceres, 2018). The parasite reduces the fecundity and lifespan of infected hosts (Auld et al., 2012). Metschnikowia is an obligate killer, meaning it must kill its host in order to transmit to a new host (Ebert, 2005); transmission spores are released into the environment after host death, after which they can be consumed by a new host, completing the parasite’s life cycle.

The experiment was run in two blocks designed to address different ecological and methodological questions. The first experiment addressed the main ecological question posed in the introduction of this manuscript. Other studies have reported adsorption of microcystins by plastics (Hyenstrand et al., 2001; Moura et al., 2022), which raised the possibility that the results of our first experiment may have been due to toxin concentrations that were lower than we intended. Therefore, in the second experiment, we evaluated whether the plastic vessels we used in the first block adsorbed microcystin-LR from the water and whether using glass vs. plastic vessels for the incubations impacted infections.

In the first experiment, we incubated fungal transmission spores for different lengths of time in filtered lake water (Pall AE filters, 1 µm pore size). The incubation times were 24 hours, 3 days, 5 days, 7 days, and 10 days. We also added two common cyanobacterial toxins, microcystin-LR and anatoxin-a, to the water during the incubations. We chose these cyanotoxins because they are commonly produced during blooms (Huisman et al., 2018) and because prior research suggested that they reduced infection prevalence (in the case of microcystin-LR) or that production of them can increase in the presence of Metschnikowia (in the case of anatoxin; Sánchez et al., 2019). Microcystin-LR is produced by some members of the genus Microcystis, which has been extensively studied due to concerns over its geographical expansion and capability of producing CHABs in both marine and freshwater ecosystems (Huisman et al., 2018). Anatoxin-a is produced by some members of the genus Anabaena. Microcystin-LR is a hepatotoxin while anatoxin-a is considered a potent neurotoxin in vertebrate models (Christensen & Khan, 2020). Both toxins are also produced by other genera of cyanobacteria such as Plantktothrix, Oscillatoria, Aphanizomenon, Cylindrospermum, and Dolichospermum, all capable of producing CHABs (Huisman et al., 2018). We also included two types of negative controls: a solvent control of 0.01% acetic acid (see toxin preparation for explanation) and a negative control with no toxin or solvent added. The incubation times were crossed factorially with the toxin treatments, as described below. After the appropriate incubation time, we carried out infection assays in which we exposed Daphnia hosts to these spores and measured infection, spore production, host reproduction, and host mortality over time.

Toxin preparation

Pure microcystin-LR standard was purchased from Cayman Chemical (Ann Arbor, MI) and suspended in 1mL of nano-pure water for a concentration of 1 mg/mL. Anatoxin-a standard was acquired from Abraxis (Warminster, PA). The anatoxin-a comes in a solution of 3:1 water and methanol and 0.01% acetic acid. We placed 1.5 mL of the anatoxin solution in a 2 mL Eppendorf tube and evaporated the methanol using an Eppendorf Vacufuge (Eppendorf, Hamburg, Germany) at 23 °C. Once evaporation had occurred, we added nanopure water to restore to the original volume/concentration. Toxins were frozen in between uses during the exposure period.

Spore preparation

Spores for the experiment were grown in vivo by infection of Daphnia dentifera (“Standard” genotype) in the laboratory. Infected animals with well-developed late-stage infections were collected from laboratory cultures and placed in 2mL Eppendorf tubes with 100-500 uL of milliQ water, then stored in the refrigerator at 4 °C. For this experiment, we used spores from animals that had been stored in the refrigerator for 2 weeks. To generate the spore slurry for experimental infections, we crushed infected animals to release spores, and then determined the density of mature ascospores using a hemocytometer (Hausser Scientific 3100, Horsham, PA, USA) and a compound microscope (Olympus BX53, Center Valley, PA, USA) at 400X magnification. Each incubation treatment was initiated with a new spore slurry (made from infected animals that were harvested live from the laboratory cultures, then stored in the refrigerator for 2 weeks) because we know that Metschnikowia spores lose infectivity over time, even in the refrigerator (Duffy & Hunsberger, 2019).

Toxin exposure to transmission spores

On the first day of the experiment (“day 1”), we initiated the longest incubation treatment (10 days) by placing 5000 mature transmission spores of Metschnikowia bicuspidata in 15 mL Falcon tubes filled with 10 mL of lake water that had been filtered through a Pall AE filter (Pall Corporation, Port Washington, NY). We then added microcystin-LR, anatoxin-a, acetic acid (negative control), or no toxin/chemical; for both microcystin-LR and anatoxin-a, we had two toxin levels: 10 and 30 µg/L. The concentrations we chose for this study are below and/or well within the range observed during natural CHABs blooms (Park et al., 1998; Pawlik-Skowrońska et al., 2004; Ibelings et al., 2005; Ha, Hidaka & Tsuno, 2009), and therefore ecologically relevant. These treatment doses are below the LC50s reported in previous Daphnia toxicology experiments for microcystins and anatoxin-a (DeMott, Zhang & Carmichael, 1991; Pawlik-Skowrońska, Toporowska & Mazur-Marzec, 2019). Even though we did not expose hosts to these toxins, we chose these concentrations because they should not cause high levels of stress and mortality in Daphnia, so any impact to pathogens could have a substantial impact on parasite-host interactions. While these concentrations are likely on the high side of what spores are likely to encounter in nature, if there is no impact of the cyanotoxins at these levels, it suggests that they are unlikely to significantly impact the free-living stages of this parasite in the water column. There were 10 replicates of each treatment (including the negative control treatment of acetic acid), with the exception of the no toxin controls (0 µg/L), which had 20 replicates; this yielded a total of 70 experimental units per incubation time treatment. The tubes with spores were left uncapped for 10 days inside a large plastic tote covered with a lid at 20 °C with a 16:8 L:D photoperiod. On day 4, the same procedure from day 1 was repeated. In this treatment, spores were incubated for 7 days at 20 °C with a 16:8 L:D photoperiod, yielding the 7-day incubation treatment. On day 6, the same procedure was used to initiate the 5-day incubation treatment. Additionally, on this day, adult Daphnia were set up so that we could use their offspring in the infection assays. Adult Daphnia of the MID37 genotype were placed in 150 mL beakers (5 adults per beaker) filled with 100 mL of filtered lake water. Beakers were placed in incubators at 20 °C with a 16:8 L:D photoperiod for 24 hours. On day 7, we collected neonates (0-24 hours old) produced from mothers that had been set up the previous day. We placed 10 neonates per beaker in 250 mL beakers with 150 mL filtered lake water for a total of 400 animals. Each beaker received 2 mg C per L of Ankistrodesmus falcatus food and was placed in incubators at 20 °C with a 16:8 L:D photoperiod. After that, 2 mg C per mL of Ankistrodesmus was added to each beaker each day until the day the experimental animals were exposed to parasites. On days 8 and 10, we used the same procedure as described above to initiate the 3- and 1-day incubation treatments, respectively.

Infection assays

On day 11, we carried out infection assays, in which we exposed Daphnia to transmission spores that had been incubated for different time lengths and with varying levels of exposure to toxins/chemicals. All Falcon tubes, containing spores with different exposure times and toxin levels, were collected and placed in a centrifuge (Sorvall ST 16, Thermo Scientific, Waltham, MA, USA) to spin down the spores. The tubes were spun at 3000 rpm for 10 min. We decanted the tubes using a 10mL pipette without disrupting the spore pellet at the bottom. In a pilot experiment, we confirmed that the original concentration of spores was recovered from vials after a 24-hour incubation followed by centrifugation. After the water was removed (which also removed the toxin), we resuspended the spores by adding 10 mL of filtered lake water and disturbing the pellet by vigorously pipetting the water in the tube. Then, we placed one 6-7-day old Daphnia in each Falcon tube and allowed the tubes to incubate again at 20 °C with 16:8 L:D photoperiod. Because we had originally placed 5000 mature transmission spores in each tube, this yielded an exposure dose of 500 spores/mL. Hosts were fed 1 mg C per mL of Ankistrodesmus on this day; using this lower level of food on the day of exposure is common in infection assays because it promotes infection. After 24 hours (that is, on day 12), each Daphnia individual was removed from the tubes with spores and placed in a 50 mL beaker filled with 30 mL filtered lake water that did not contain spores (one animal per beaker). Animals were fed 2 mg/L C of Ankistrodesmus falcatus ad libidum for the rest of the experiment (20 days post-infection, 32 days from day 1 of the entire experiment).

During those 20 days, we tracked mortality in each of the beakers five days per week (Mondays through Fridays). Water changes were done twice a week; during these, we counted offspring in each beaker; offspring were removed from beakers and discarded. Any animals that died during the trial were placed in 2 mL Eppendorf tubes with 100 µL of nanopure water and stored at 4 °C for later spore counts. At the end of the experiment (20 days post-infection, 32 days from day 1 of the entire experiment), any remaining live animals (130 total) were placed in a 2mL Eppendorf tube with 100 µL of nanopure water. Animals were then ground to release spores, and spores in the ascus stage were counted using a hemocytometer under a compound microscope at 400X magnification.

In the second experiment, we incubated fungal transmission spores using glass conical tubes and plastic falcon tubes. For this experiment, we used 24-hour & 3-day incubation times. The primary concern that prompted this experiment is that, in a prior study, microcystin-LR was found to adsorb to plastic (Hyenstrand et al., 2001). Therefore, in this experiment, we focused on microcystin-LR, using the same concentrations as in the first experiment: 30 µg/L, 10 µg/L and a control with no toxin. In total there were nine treatments in this experiment (Figure S1). There were three microcystin-LR concentrations and two incubation lengths, crossed factorially. Most of the incubations were done in glass tubes. In addition, for the 24-hr incubation lengths in the 30 µg/L microcystin treatment, we also factorially crossed vessel type (glass vs. plastic tubes, so that we could test for an effect of vessel type) and presence/absence of spores (in order to compare the level of microcystin-LR in glass vs. plastic tubes, which was done using water from the spore-free incubations). 

This second experiment was run in a similar manner to the first experiment. Spores were incubated with their corresponding toxin treatment for 24 hours or 3 days. We focused on these shorter incubation treatments for the following reasons. First, in the earlier study, the authors detected adsorption rapidly (within seconds); thus, this should have been ample time for adsorption to occur. Second, if microcystin-LR adsorbed to the plastic we used in our experiment and reduces infectivity, we would expect to see a reduction in the infectiousness of spores incubated in glass vs. plastic. Thus, we would best be able to detect this at the incubation lengths that had yielded the highest infection levels (24 hours and 3 days).

Treatments with spores had spores added to them in a similar manner to what was described for the first experiment; for treatments with no spores, we added toxin to the appropriate vessel with filtered lake water but no spores. After the incubation period had elapsed, we centrifuged tubes to spin spores down to the bottom of the tubes; for consistency, we spun all tubes, regardless of whether they were from a treatment with spores present or absent. After centrifugation, we collected 1mL water sample from each tube; we placed these samples in 2mL glass vials and stored them at -80F for later quantification of toxin concentration in the water. The rest of the water was decanted without disturbing the spore pellet in the bottom. After spores were resuspended in new filtered lake water, 6–7-day old Daphnia dentifera (MID37 clone) were placed in each tube and allowed to incubate with spores (or no spores) for 24 hours to allow time for infection. After 24 hours, all animals were removed from tubes and placed in 50mL beakers with 30mL of filtered lake water. Animals were then fed ad-libitum for 20 days. Animals that died within 20 days of exposure were preserved in 2mL Eppendorf tubes with 100 µL of milliQ water and stored at 4C. At 20 days post-exposure, any remaining live animals were placed in a 2mL Eppendorf tube with 100 µL of nanopure water, then stored at 4C. Later, animals were removed from cold storage and ground to release spores; spores in the ascus stage were counted using a hemocytometer under a compound microscope at 400X magnification.

Data analysis

For all our models, we analyzed data from the microcystin-LR and anatoxin-a treatments separately. This means that for each analysis described below, there was one performed for the microcystin-LR relevant data and another for the anatoxin-a data. The same no-toxin control data (0 µg/L; 20 replicates per incubation time) were used for the two sets of data (microcystin-LR and anatoxin-a). When analyzing the anatoxin-a data, the acetic acid treatments were included in the analyses (as an additional negative control) and were treated in our analyses as a low concentration (0.01%); statistical analysis did not find differences between the no toxin controls and the acetic acid controls (data not shown). Prior to analyses, the data were checked for normality using the Shapiro-Wilk test. Data that did not meet normality were analyzed using appropriate family error distribution link functions and checking for overdispersion. All analyses were carried out in R Studio Version 1.2.1335 using the stats v3.6.1 package.

We assessed environmental effects on the parasite’s ability to infect by comparing differences in the number of animals that developed terminal infections (that is, infections that produced asci (Stewart Merrill & Cáceres, 2018)). In this analysis, our response variable had two outcomes (terminal infection or not). We performed a generalized linear model (GLM) with incubation time and toxin concentration as explanatory variables, using a binomial family error distribution.

We also evaluated if toxin concentration and incubation time (that is, the time spent as a transmission spore in the water) affected the number of mature transmission spores produced per infected host individual. For this analysis, we ran a GLM; here the number of spores was our response variable and, similarly to the analysis of infections, toxin concentration and incubation time were used as the explanatory variables. In this model, we used a Gaussian error distribution.

To evaluate the effects of incubation time and toxin exposure on the parasite’s virulence, we measured host offspring production, host survival over the 20-day experiment, and the proportion of hosts who died within 20 days of parasite exposure. For these analyses, we ran GLMs with toxin concentration and time of exposure as explanatory variables; for the two former response variables—the number of offspring each host individual produced and the number of days each host survived (up to 20 days post-infection)—we used a Poisson family error distribution. For the proportion of dead hosts at the end of the experiment, we used a binomial family distribution.

We ran two linear models analyzing the relationship between the proportion of infected individuals and the mean number of spores produced at the end of infection. In the first, we averaged across the different toxin exposure treatments, yielding one value for each incubation time (n = 5). In the second, we averaged within the toxin treatments, yielding five values per toxin treatment (one per incubation time treatment, total n = 20).

For the second set of experiments, we evaluated the data in two ways. First, we evaluated the same response variables as explained above for the first experiment and evaluated whether the results were qualitatively similar or different. Second, we merged the data sets from the first and second experiments, using data only from the microcystin treatments, and, in the case of the data from the second experiment, we used both plastic and glass data adding "vessel" as part of the model. We then used a generalized linear mixed model (GLMM) with the same set-up as the first experiment in terms of response and treatment variables and using “first” or “second” experiment as a random block variable.

Finally, using data from the second experiment, we also evaluated whether dissolved toxin concentration was different when using different exposure vessels after 24 hours by bootstrapping, and whether there were differences in infection prevalence, spore production and offspring reproduction when using different vessels for the exposure of spores. More details on these analyses and results can be found in the supplement. Results were qualitatively the same regardless of whether exposures were done in glass vs. plastic and toxin levels did not significantly differ between the two vessel types (see supplement), leading us to conclude that the effects seen in our first experiment were not altered by the use of plastic tubes. Therefore, in the results in the main text, we present only the results from experiment 1, because this had all incubation lengths and both toxins. 

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